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MAHARASHTRA COLLEGE OF PHARMACY

APPLICATION OF cDNA MICROARRAY TECHNOLOGY IN CARCINOGEN IDENTIFICATION Page 1

INTRODUTION

A DNA microarray is a multiplex technology used in molecular biology

and in medicine.

Microarray technology evolved from southern blotting where

fragmented DNA is attached to a subtrateand then probed with a known

gene or fragment .

The basic principle underlying microarray technology is that

complementary nucleic acids will hybridize. Hybridization provides exquisite

selectivityof complementary stranded nucleic acids, with high sensitivityand

specificity. In the traditional techniques, in which radioactivelabeling

materials are usually used, the simultaneous hybridizationof test and

reference samples is impossible

DNA microarray technology has empowered the scientific community

to understanding the fundamental aspect underlying the growth and

development of life as well as to explore the genetic cause of anomalities

occurring in the functioning of the human body.

A typical microarray experiment involves the hybridization of an mRNA

molecule to the DNA template from which it is originated.

The advantages of the cDNA microarray comparedwith the

oligonucleotide array have been thought to includeless susceptibility and

higher specificity due to the longersequences of the targets .However,

cDNAmay contain repetitive sequences that are often observed invarious

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3. Comparative Genomic Hybridization

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Principal of micro array technology;

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The basic principle underlying micro array technology is thatcomplementary

nucleic acids will hybridize. Hybridization provides exquisite selectivityof

complementary stranded nucleic acids, with high sensitivityand specificity. In

the traditional techniques, in which radioactive

labeling materials are usually

used, the simultaneous hybridizationof test and reference samples is

impossible

In micro array-basedtechnologies, the solid surface, such as a glass

slide, containshundreds to thousands of immobilized DNA (targets) spots

whichcan be simultaneously hybridized with two samples (probes) labeledwith

different fluorescent

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Applications include:

Technology or

ApplicationSynopsis

Gene expression

profiling

used to identify genes whose expression is

changed in response to pathogens or other

organisms by comparing gene expression in

infected to that in uninfected cells or tissues.

Chromatin

immunoprecipitation

on Chip

DNA sequences bound to a particular protein can

be isolated by immunoprecipitating that protein

these fragments can be then hybridized to a

microarray allowing the determination of protein

binding site occupancy throughout the genome.

SNP detection

microarrays make use of SNP detection, including

Genotyping, forensic analysis, measuring

predisposition to disease, identifying drug-

candidates, evaluating germline mutations in

individuals or somatic mutations in cancers,

assessing loss of heterozygosity, or genetic

linkage analysis.

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Gene expression:The sequence of a gene does not give information about its activity. A

gene must be transcribed into mRNA to be expressed (or turned ON). The

level of expression (or activity) of a gene can therefore be studied by

measuring how much corresponding mRNA is made (transcribed) from a

particular gene.

The sequence of a gene is complementary to the sequence of its mRNA.

Therefore the mRNA can base pair (or hybridize) to the DNA strand it was

copied from (template strand). Scientists study gene expression by

measuring the amount of mRNA for different genes.

ALL of our cells contain the exact same DNA sequence. All cells arose

from a single cell (the egg). However different type of cells in the body

expresses different sets of genes. For example, a liver cell expresses some

of the same genes expressed in lung cells or skin cells but also expresses

genes that no other cell type expresses (liver-specific genes). As a result

liver cells look different from other cells in the body and do things that no

other cell can do.

Microarrays are simply a method for visualizing which genes are likelyto be used in a particular tissue at a particular time under a particular set

of conditions. The output of a microarray experiment is called a e gene

expressionprofile.

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Drug Target:

DNA microarray which allows the monitoring of gene expression level of

thousands of genes simultaneously played a significant role in drug discovery.

Animals may not be fully predictive of the response in human due to

species variation. The pre-clinical studies in animals do not always detect

drugs that prove to be toxic in human. Ex. tacrine, used to treat patients

with Alzheimerds disease include hepatotoxicity by the elevation of liver

transminase in human, but there was no evidence of liver toxicity in mice

rats ordogs during pre-clinical development. Drug-inducedhepatotoxicity

andliverdamage cause significant morbidity andmortality andrepresent a

major challenge inthe area of drugdevelopment anddesign.

Microarray databases are built on the expression data from well

definedmarketedpharmaceuticals, classical chemical toxicants andfew

proprietary drugs. These compounds have well definedpharmacological action

andsome are rat andhuman specific, which result in data bases that are

useful for predicting toxicity duringdevelopment of toxicity.

Microarray can be usedto screen for changes in gene expression

following exposure of tumour cells to drugs either in culture or in patients

following treatment. Studies on this type may provide information on the

precise mechanism of action of the drug or couldlead to the identification

of early markers of drugresponse. For predicting the longer term clinical

response to drug treatment, microarry couldalso yieldinformation about

possible side effects, andidentify markers that, in clinical context, couldbe

usedto predicts possible adverse effect events.

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App

lication of Microaray:

y Use of Microarrays in Tissue Engineering, Phenotype Analysis, andMonitoring

y Microarray Application to Oral Biology and Mediciney Gene discoveryy Drug discovery and developmenty Toxicological researchy Tumour classificationy Tumour growgh and progressiony Disease diagnosisy Detection, identification, and characterization of food-borne

pathogens

y DNA microarrays are being analyzed to understand the innate immunesystem, including the cellular response to various bacterialendotoxins

y DNA microarraysare now being used to identify other genes that maybe regulatedin response to endotoxins, such as LPS, with the

ultimate hopeof providing new drug targets for sepsis With the use

of human umbilical vein endothelial cells as a model, investigators

foundvarious genes that appear to be regulated in response toLPS. Many

of the genes identified confirm already well-establishedLPS-

regulated genes, whereas a few may represent new gene targets

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y Microarray technology has also been used to monitor changes inmetabolic processes including aging

Challenges:The limited availability or complete absence of in vivo human data adds

additional challenges to the analysis of microarrays using comparative

genomics. In such an instance, the effects observed using in vivo animal

models and in vitro human data need to be projected to the effects in

humans. Hence, a challenge is to extrapolate from these limited studies

whole-body human effects while accounting for potentially not well-known

or wellunderstood nonlinearities.

y Lack of Appropriate Arraysy Lack of Comparable Studiesy Applications will encompass defining gene function;y Inferring functional networks and pathways;y Understanding how variation is distributed among individuals,

populations, and species;

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y Adams JC, Watt FM (1993). Regulation of development anddifferentiation by the extracellular matrix. Aharoni A, Keizer LCP,

Bouwmeester HJ, Sun Z, Huerta MA, Verhoeven HA, et al. (2000).

Identification of the SAAT gene involved in strawberry flavor

biogenesis by use of DNA microarrays.

y Alizadeh AA, Eisen MB, Davis ER, Ma C, Lossos IS, Rosenwald A, etal. (2000). Distinct types of diffuse large B-cell lymphoma identified

by gene expression profiling.

y Ashkenas J, Muschler J, Bissell MJ (1996). The extracellular matrix inepithelial biology: shared molecules and common themes in distant phyla.

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